Curcumin Increased the Sensitivity of Non-Small-Cell Lung Cancer to Cisplatin through the Endoplasmic Reticulum Stress Pathway

Objective Non-small-cell lung cancer (NSCLC) is one of the most lethal cancers. Although cisplatin-based chemotherapies have been regarded as a promising treatment approach, cisplatin resistance still remains one of the major clinical challenges. Curcumin, a naturally occurring polyphenol, has been proved to increase chemotherapeutic efficiency of NSCLC cells. However, the role of curcumin in cisplatin-resistant NSCLC cells has been rarely investigated. This study aims to investigate whether curcumin enhances cisplatin sensitivity of human NSCLC cells and its underlying mechanisms. Method A549/DDP and H1299/DDP cells were treated by DDP or/and curcumin before cell viability, and apoptosis were determined by using a CCK-8 assay and flow cytometer. The expressions of apoptosis and ER stress-related proteins, including cleaved caspase-3, cleaved PARP, CHOP, GRP78, XBP-1, ATF6, and caspase-4, were measured by the qPCR and western blotting. After cotreatment by DDP and curcumin, A549/DDP and H1299/DDP cells were further treated by the ER stress inhibitor, salubrinal (20 μm), after which the cell apoptosis and viability were detected. Result Treatment by DDP and curcumin can substantially decrease cell viability, while can increase the cell apoptosis rate, elevate mRNA and protein expressions of apoptosis and ER stress-related proteins, compared with cells treated by DDP or curcumin alone. Salubrinal treatment can counteract the suppressive effect of DDP and curcumin on cell viability and decrease the cell apoptosis of A549/DDP and H1299/DDP cells. Conclusion Curcumin can increase the sensitivity of NSCLC to cisplatin through an ER stress pathway and thus can be served as one of the molecular targets for overcoming the cisplatin resistance.


Introduction
Lung cancer is one of the most lethal cancers worldwide, which can be clinically classified into non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC) [1]. e former accounts for approximately 85% of all lung cancer cases [2,3]. Platinum agents, including cisplatin (DDP), are considered to be one of the reference drugs for first-line treatment of NSCLC [4]. Although cisplatin-based chemotherapies proved to have a remarkable curative effect on NSCLC patients, cisplatin resistance developed by NSCLC cells continues to be a major challenge in clinic [5,6]. e strategy of increasing a cisplatin dose can potentially overcome drug resistance but also leads to increased possibility to develop life-threatening adverse side effects. us, searching for strategies to increase the cisplatin sensitivity of NSCLC cells will be highly desirable to overcome drug resistance. e endoplasmic reticulum (ER) is the first intracellular compartment of the secretory pathway, which regulates calcium homeostasis, lipid biosynthesis, and protein productive folding and quality control. However, under certain cytotoxic conditions, such as hypoxia and nutrient deprivation, protein misfolding occurs via disruption of proper ER function, leading to unfolded proteins accumulating and aggregating in the ER, known as ER stress [7][8][9][10][11][12]. ER stress was associated with anticancer drug resistance via unfolded protein response [10,13].
Curcumin extracted from the plant of Curcuma longa was reported to possess antitumor activity through regulating oncogene expression, cell cycle regulation, apoptosis, tumorigenesis, and metastasis PMID: 34885693. Recently, curcumin was found to promote chemotherapeutic efficiency in various cancers and diseases [9,[14][15][16][17]. For instance, Zou et al. reported that curcumin increases breast cancer cell sensitivity to cisplatin by decreasing FEN1 expression [9]. Lu et al. found that curcumin can increase the sensitivity of paclitaxel-resistant NSCLC cells to paclitaxel through microRNA-30c-mediated MTA1 reduction [16]. So far, the possible effect of curcumin on cisplatin sensitivity of lung cancer cell has not been well investigated and worthy further explorations. Recently, curcumin was found to increase effects of irinotecan through mediating the ER stress pathway in colorectal cancer cells [18,19]. Misra et al. found that curcumin regulates ER stress through cAMP responsive element-binding protein H [20]. So far, the implication of ER stress in curcumin-attenuated drug resistance of lung cancer cells has been rarely investigated.
Inspired by previous studies, this study intends to investigate whether the ER stress pathway is the key factor for curcumin-increased NSCLC sensitivity to cisplatin, with the expectation to provide a new strategy and molecular target for overcoming the cisplatin resistance in lung cancer cells.

Materials and Methods
2.1. Cell Culture. Human lung cancer cell lines A549 and H1299 originated from the American Type Culture Collection were cultured in the RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS, Gibco) in a humidified atmosphere containing 5% CO 2 at 37°C. To establish cisplatin-resistant A549 and H1299 cell lines, A549 and H1299 cells were firstly treated with a 0.5 μM of cisplatin (DDP, Sigma) and then were treated with increased concentrations of DDP in a stepwise manner during cell passage. To maintain the drug-resistant phenotype, DDP (with final concentration of 1.5 μM) was added into the culture media for A549/DDP and H1299/DDP cells.

Cell Proliferation
Detected by the CCK-8 Assay. A549, A549/DDP, H1299 and, H12999/DDP cells were seeded in 96-well plates at the density of 5 × 10 3 /well. After the cell culture for 12 h, cells were then treated with various concentrations of curcumin (1.25, 2.5, 5, 10, and 20 μg/mL) or/ and cisplatin (2, 4, 8, 10, and 12 μg/mL). A549/DDP and H12999/DDP cells were treated by 2 μg/mL DDP, 2.5 μg/mL curcumin or DDP + curcumin, which were, respectively, named as the DDP group, curcumin group, and curcumin + DDP group. Equal volume of PBS was used in the control group. After cell incubation for 48 h, the 10 μL CCK-8 (Sigma-Aldrich, USA) reagent was added to each well for incubation of 2 h. e absorbance at 450 nm in each well was determined by using a microplate reader.

Cell Apoptosis Detected by the Flow Cytometry Assay.
e cell apoptosis rate was detected by using the Annexin V-FITC/PI kit (BD Biosciences) based on the instructions. Cells digested by pancreatin were collected after centrifugation, and after that, cells were then resuspended in a binding buffer before further incubation with Annexin V-FITC and PI for 15 min. Cell apoptosis was calculated using a flow cytometry method. All experiments were conducted 3 times.

Protein Expression Levels by the Western Blotting Assay.
e expression levels of protein-activating transcription factor 6 (ATF6), C/EBP homology protein (CHOP), and caspase-4 were detected by the western blotting assay. e logarithmic growth cells were inoculated on 6-well plates at the density of 1 × 10 5 cells/well. Each well was added with corresponding concentrations of curcumin or cisplatin solution in a serum-containing medium. After the cells were lysed, we collected the supernatant and then extracted the cytosolic protein or nuclear protein. e protein centrifugation liquid was transferred into the PVDF membrane after SDS-PAGE vertical electrophoresis. Electrophoretic analysis of equivalent protein lysates was performed.

Real-Time Quantitative PCR Assay Detected Expression of mRNA.
Total RNA was isolated from cells using the TRI-ZOL reagent and then subjected to reverse transcription using the RT kit (TaKaRa, Tokyo, Japan) based on the instructions specified in the kit. e Biosystems 7300 real-time PCR system (ABI, Foster City, CA, USA) was used for PCR using SYBR GreenMix (Takara). ree duplicates were set for each reaction of PCR. Data analysis was determined using the 2 −ΔΔCt method [21]: the ΔΔ Ct � experimental group (Ct target gene −Ct internal control )− the control group (Ct target gene −Ct internal control ). GAPDH was used as the internal control, and primer sequences are listed in Table 1.

Statistical Analysis.
GraphPad Prism7 was used for data analysis. Experiment data were presented as the mean-± standard deviation (SD). Student's t-test was used to analyze statistical significance between pairwise groups, while comparisons among groups were analyzed using oneway analysis of the variance, with Tukey's multiple comparisons for post hoc analysis. Results with p < 0.05 and p < 0.01 are considered statistically significant ( * ) and ( * * ), respectively.

Curcumin Decreases Cell Viability of A549/DDP and H1299/DDP Cells.
e effect of various concentrations of DDP on A549, H1299, A549/DDP, and H1299/DDP cells was assessed by the CCK-8 assay. e results demonstrated that DDP can significantly decrease cell viability of A549 and H1299 cells in a concentration dependent manner, compared with those in A549/DDP and H1299/DDP cells (Figures 1(a) and 1(b), * P < 0.05). e medium inhibitory concentration (IC50) of DDP on A549 and H1299 cells was 4 μg/mL, while that for A549/DDP and H1299/DDP cells was 12 μg/mL, suggesting A549/DDP and H1299/DDP cells had obvious resistance to DDP, which was similar to the findings in a previous study [22]. en, the cytotoxicity of curcumin against A549/DDP and H1299/DDP cells was assessed by the CCK-8 assay. As shown in Figures 1(c)and 1(d), curcumin can suppress the viability of A549/DDP and H1299/DDP cells in a concentration-dependent way. Specifically, cell viability of A549/DDP and H1299/DDP cells was reduced to approximately 48% after treatment with 10 μg/mL curcumin for 48 h. us, the IC50 of curcumin against A549/DDP and H1299/DDP cells was 10 μg/mL. In addition, the maximum nonlethal concentration of curcumin against A549/DDP and H1299/DDP cells was 2.5 μg/ mL. ese results showed that curcumin can significantly decrease cell viability of drug-resistant NSCLC cells.

Curcumin Increases the Sensitivity of Resistant NSCLC
Cells to DDP. Although curcumin can suppress cell viability of A549/DDP and H1299/DDP cells, but whether curcumin can improve the sensitivity of A549/DDP and H1299/DDP cells to DDP remains unknown. We assessed the cell viability of A549/DDP and H1299/DDP cells after cotreatment by various concentrations of DDP and curcumin (2.5 μg/mL).
e CCK-8 assay showed compared with the DDP group that the cell viability of A549/DDP and H1299/DDP cells in the DDP + curcumin group was significantly decreased (Figures 2(a) and 2(b), * P < 0.05), and the concentration of DDP for IC50 in the DDP + curcumin group was 2 μg/mL. e combined effect of DDP (2 μg/mL) and curcumin (2.5 μg/mL) on A549/DDP and H1299/DDP cells was determined by the CCK-8 assay, which demonstrated that the cell viability of A549/DDP and H1299/DDP cells in the DDP + curcumin group was decreased significantly compared with the curcumin group and the DDP group (Figure 2(c), & P < 0.05). Flow cytometry showed that the cell apoptosis rate in the curcumin group and the DDP group was elevated compared with that in the control group ( # P < 0.05) but decreased when compared with that in the DDP + curcumin group. In addition to that, the promotive effect on cell apoptosis in the DDP + curcumin group was increased by 50% compared with that in the DDP group (Figure 2(d), & P < 0.05). Measurement on expressions of apoptotic-related proteins (cleaved caspase-3 and cleaved PARP) by western blotting showed that the expressions of cleaved caspase-3 and cleaved PARP in the curcumin group and the DDP group were elevated compared with those in the control group ( # P < 0.05) but decreased compared with those in the DDP + curcumin group (Figures 2(e)and2(f ), # P < 0.05). ese results showed that curcumin can increase the sensitivity of resistant NSCLC cells to DDP, decrease cell viability, and promote cell apoptosis.

Curcumin Regulates the Expressions of ER Stress-Related
Proteins. Evidence in previous studies showed that ER stress is closely related to drug resistance of tumor cells [10,23]. erefore, we detected the expressions of ER stress-related proteins, including CHOP, GRP78, XBP-1, ATF6, and caspase-4 in A549/DDP and H1299/DDP cells. e expressions of ER stress-related proteins in the control group showed no significant difference with those in the curcumin group (Figures 3(a)-3(d)), but those expressions were elevated in the DDP group when compared with the control group (Figures 3(a)-3(d)). After the cells were cotreated by DDP and curcumin, the expressions of CHOP, GRP78, XBP-1, ATF6, and caspase-4 in the curcumin + DDP group increased significantly than those in the either curcumin or DDP group (Figures 3(a)-3(d)). ese results showed that

Genes
Sequences (5′ end to 3′ end)  (Figures 4(a)-4(d)). Subsequently, we measured the effect of salubrinal on the cell viability and apoptosis rate. CCK-8 and flow cytometry showed that compared with cells in the curcumin + DDP + DMSO group, A549/DDP and H1299/ DDP cells in the curcumin + DDP + salubrinal group had elevated cell viability and decreased the apoptosis rate (Figures 4(e)and4(f )). Western blotting also demonstrated that the expressions of cleaved caspase-3 and cleaved PARP in the curcumin + DDP + salubrinal group were suppressed compared with those in the curcumin + DDP + DMSO group (Figure 4(g)). e working mechanism of curcumin increasing DDP sensitivity of resistant NSCLC cells through ER stress. DDP, cisplatin; NSCLC, non-small-cell lung cancer; and ER stress, endoplasmic reticulum stress.

Discussion
DDP is considered to be one of the most promising chemotherapy drugs broadly used for various types of human epithelial cancers, including ovarian carcinoma, lung carcinoma, breast carcinoma, and head and neck carcinoma [25][26][27]. Nevertheless, DDP resistance is a major challenge for cisplatin-based chemotherapy. Cisplatin-resistant cells exhibit decreased intracellular DDP accumulation due to enhanced efflux and reduced influx [28]. DDP can also be inactivated by sulfur-containing macromolecules, including metallothionein and glutathione [29]. Exploration of novel strategies to increase the sensitivity of cancer cell to DDP for overcoming the drug resistance will be highly desirable. In this study, we demonstrated that the exposure of cancer cells to sublethal doses of curcumin could promote DDP chemotherapeutic efficiency against NSCLC cells, in which the ER stress pathway mediated by the CHOP, ATF6, and caspase-4 plays an important role.
Curcumin, as a cancer chemosensitizing agent, can effectively reduce resistance to many chemotherapy drugs, including DDP, mitomycin C, and paclitaxel, in a wide variety of tumor cells [7,8,10,15,16]. Due to its chemosensitizing effect, a combination of cisplatin with curcumin   [30,31]. In addition, the DDP-chemosensitizing effects of curcumin against NSCLC cell viability and apoptosis were demonstrated by using the CCK-8 assay and the flow cytometer. ER stress causes apoptosis through following mechanisms, including expression of CHOP, activation of ER resident caspase, and induction of the ASK1-JNK pathway [32]. In particular, CHOP is the first molecule identified to mediate ER stress-induced apoptosis. e unfolding protein response of ER stress exceeds the threshold, and damaged cells are committed to apoptosis through the ATF6-mediated GADD153 signaling pathway [33]. Recently, ER stress is one of the molecular mechanisms responsible for curcumininduced apoptosis [34,35]. In this work, to validate the hypothesis that curcumin increased the sensitivity of NSCLC to DDP through the ER stress pathway, we detected the expression levels of ER stress-related proteins (CHOP, ATF6, and caspase-4). As expected, combined treatment of curcumin and cisplatin significantly increased protein and RNA levels of CHOP, ATF6, and caspase-4 in comparison with curcumin or cisplatin alone. In addition to that, the combination of curcumin and DDP on A549/DDP and H1299/ DDP cells led to the decreased cell viability and elevated apoptosis rate, indicating curcumin increased DDP sensitivity through regulating ER stress. is result was further supported by the elevated cell viability and decreased apoptosis rate in A549/DDP and H1299/DDP cells treated with combination of curcumin, DDP, and ER stress inhibitor salubrinal.

Evidence-Based Complementary and Alternative Medicine
In conclusion, the current study revealed that the ER stress pathway is associated with acquired DDP resistance in NSCLC cells and curcumin can enhance the chemosensitizing effect of NSCLC cells by targeting the ER stress pathway. Taken together, this study demonstrated curcumin can increase DDP sensitivity of NSCLC cells through mediating the ER stress pathway. A better understanding on the drug resistance of cancer cell can facilitate the solution   against drug resistance. e results of this study proposed a possible mechanism of curcumin improving DDP sensitivity in NSCLC cells and may shed a little light on developing the molecular targets in the ER stress pathway to overcome the DDP resistance in NSCLC.

Data Availability
e authors can make data available on request through a data access committee and institutional review board. In addition, all the data can also be obtained from author Lile Wang.

Conflicts of Interest
e authors declare that they have no conflicts of interest.  Figure 4: Curcumin increases the sensitivity to DDP-resistant NSCLC cells through regulating ER stress. After A549/DDP and H1299/DDP cells were cotreated with DDP and curcumin, salubrinal was used as an ER stress inhibitor to treat A549/DDP and H1299/DDP cells. (a-d) e expressions of CHOP, GRP78, XBP-1, ATF6, and caspase-4 were detected by qPCR and western blotting. (e) CCK-8 was used to detect cell viability; (f ) the cell apoptosis rate was detected by flow cytometry. (g) Western blotting was used to measure the expressions of cleaved caspase-3 and cleaved PARP. Experiment data are presented as the mean ± standard deviation (SD), each experiment was conducted 3 times. * P < 0.05, when compared with the curcumin + DDP + DMSO group; DDP, cisplatin; NSCLC, non-small-cell lung cancer; ER stress, endoplasmic reticulum stress.